Mir142 loss unlocks IDH2R140-dependent leukemogenesis through antagonistic regulation of HOX genes.

AML is a genetically heterogeneous disease and understanding how different co-occurring mutations cooperate to drive leukemogenesis will be crucial for improving diagnostic and therapeutic options for patients. MIR142 mutations have been recurrently detected in IDH-mutated AML samples. Here, we have used a mouse model to investigate the interaction between these two mutations and demonstrate a striking synergy between Mir142 loss-of-function and IDH2R140Q, with only recipients of double mutant cells succumbing to leukemia. Transcriptomic analysis of the non-leukemic single and leukemic double mutant progenitors, isolated from these mice, suggested a novel mechanism of cooperation whereby Mir142 loss-of-function counteracts aberrant silencing of Hoxa cluster genes by IDH2R140Q. Our analysis suggests that IDH2R140Q is an incoherent oncogene, with both positive and negative impacts on leukemogenesis, which requires the action of cooperating mutations to alleviate repression of Hoxa genes in order to advance to leukemia. This model, therefore, provides a compelling rationale for understanding how different mutations cooperate to drive leukemogenesis and the context-dependent effects of oncogenic mutations.

Author Correction: Osmotic modulation of chromatin impacts on efficiency and kinetics of cell fate modulation.

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Osmotic modulation of chromatin impacts on efficiency and kinetics of cell fate modulation.

Chromatin structure is a major regulator of transcription and gene expression. Herein we explore the use of osmotic modulation to modify the chromatin structure and reprogram gene expression. In this study we use the extracellular osmotic pressure as a chromatin structure and transcriptional modulator. Hyposmotic modulation promotes chromatin loosening and induces changes in RNA polymerase II (Pol II) activity. The chromatin decondensation opens space for higher amounts of DNA engaged RNA Pol II. Hyposmotic modulation constitutes an alternative route to manipulate cell fate decisions. This technology was tested in model protocols of induced pluripotency and transdifferentiation in cells growing in suspension and adherent to substrates, CD34+ umbilical-cord-blood (UCB), fibroblasts and B-cells. The efficiency and kinetics of these cell fate modulation processes were improved by transient hyposmotic modulation of the cell environment.

Expression of the fetal hematopoiesis regulator FEV indicates leukemias of prenatal origin.

The origin of cancers is associated with etiology as well as therapeutics. Several studies reveal that malignancies in children can originate in utero. However, a diagnostic approach to distinguish between cancers initiated pre- or postnatally is absent. Here we identified a transcriptional factor FEV (fifth Ewing variant) that was expressed in fetal hematopoietic cells and became silent after birth. We characterized that FEV was essential for the self-renewal of hematopoietic stem cells (HSCs). We next found that FEV was expressed in most infant leukemia samples, but seldom in adult samples, in accord with the known prenatal origins of the former. We further determined the majority of pediatric acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) were FEV positive. Moreover, FEV knockdown markedly impaired the leukemia-propagating ability of leukemic stem cells. We therefore identified FEV is unique to fetal HSCs and stably expressed in leukemic cells of prenatal origin. It may also provide a tractable therapeutic target.

Stem cell programs are retained in human leukemic lymphoblasts.

Leukemic lymphoblasts within different immunophenotypic populations possess stem cell properties. However, whether or not the self-renewal program is retained from stem cells or conferred on progenitors by leukemogenic molecules remains unknown. We have addressed the issue in the context of TEL-AML1-associated acute lymphoblastic leukemia (ALL) by profiling a refined program edited from genes essential for self-renewal of hematopoietic stem cells and B-cell development. Bioinformatic analysis shows that ALL populations are loosely clustered and close to the normal population that contains stem and primitive progenitor cells. This finding indicates that immunophenotypes do not reflect maturation stages in ALL and that the self-renewal program may be retained from stem cells. Results of assessing 'first hit' function of TEL-AML1 in different populations of normal cells demonstrate the molecular model. Therefore, the current study shows a leukemogenic scenario of human ALL in which programs of stem cells are sustained in distinct fractions by leukemogenic mutations.

Surgical Treatment of 55 Patients with Pressure Ulcers at the Department of Plastic and Reconstructive Surgery Kosovo during the Period 2000-2010: A Retrospective Study.

Objective. The objective of this study is to determine the incidence of PUs, the distribution of PUs, common injuries contributing to the occurrence of PUs in patients admitted to the Department of Plastic and Reconstructive Surgery Kosovo for surgical interventions of PUs, localization of PUs in body, the topical treatment of pressure ulcers before surgical intervention, the methods of surgical interventions, number of surgical interventions, duration of treatment, complications, and mortality. Materials and Methods. This study includes 55 patients with PUs treated surgically in 2000-2010 period in the Department of Plastic and Reconstructive Surgery Kosovo. The data were collected and analyzed from the archives and protocols of the University Clinical Center of Kosovo. Data processing was done with the statistical package In Stat 3. From statistical parameters arithmetic median and standard deviation were calculated. Data testing is done with χ (2)-test and the difference is significant if P < 0.05. Conclusion. Despite preventive measures against PUs, the incidence of Pus remains high.

Differential contributions of haematopoietic stem cells to foetal and adult haematopoiesis: insights from functional analysis of transcriptional regulators.

An increasing number of molecules have been identified as candidate regulators of stem cell fates through their involvement in leukaemia or via post-genomic gene discovery approaches. A full understanding of the function of these molecules requires (1) detailed knowledge of the gene networks in which they participate and (2) an appreciation of how these networks vary as cells progress through the haematopoietic cell hierarchy. An additional layer of complexity is added by the occurrence of different haematopoietic cell hierarchies at different stages of ontogeny. Beyond these issues of cell context dependence, it is important from a mechanistic point of view to define the particular cell fate pathway impacted by any given regulator. Herein, we advance the notion that haematopoietic stem cells (HSC), which sustain haematopoiesis throughout adult life and are specified in foetal life, have a minimal or late contribution to foetal haematopoiesis but instead largely proliferate during the foetal period. In light of this notion, we revisit published data on mouse knockouts of haematopoietically-affiliated transcription factors highlighting novel insights that may be gained from taking such a view.

Acute promyelocytic leukemia: where does it stem from?

A fundamental issue in cancer biology is the identification of the target cell in which the causative molecular lesion arises. Acute myeloid leukemia (AML) is thought to reflect the transformation of a primitive stem cell compartment. The resultant 'cancer stem cells' comprise only a minor portion of the leukemic clone but give rise through differentiation to more committed progenitors as well as differentiated blasts that constitute the bulk of the tumor. The maintenance of the leukemic clone is dependent on the self-renewal capacity of the cancer stem cell compartment, which is revealed by its ability to re-initiate leukemia in a transplant setting. The cellular basis of acute promyelocytic leukemia (APL) is however less clear. APL has traditionally been considered to be the most differentiated form of AML and to arise from a committed myeloid progenitor. Here we review apparently conflicting evidence pertaining to the cellular origins of APL and propose that this leukemia may originate in more than one cellular compartment. This view could account for many apparent inconsistencies in the literature to date. An understanding of the nature of the target cell involved in transformation of APL has important implications for biological mechanism and for clinical treatment.

Lineage restriction of the RARalpha gene expression in myeloid differentiation.

To better understand the role of retinoids in myelopoiesis, expression of the retinoid receptor genes (retinoic acid receptors [RARs] and retinoid X receptors [RXRs]) were examined during differentiation of factor-dependent cell-Paterson (FDCP)-mixA4 murine progenitor cells. The major receptor expressed in undifferentiated A4 cells was RARalpha (primarily the RARalpha1 isoform). Following induction of myelomonocytic differentiation with granulocyte and granulocyte-macrophage colony-stimulating factors, a dramatic increase in RARalpha expression (particularly the RARalpha2 isoform) was seen. In contrast, expression of both RARalpha isoforms was rapidly extinguished upon induction of erythroid differentiation with erythropoeitin (EPO). A modest induction of RXRalpha expression was seen, particularly during differentiation in the myelomonocytic lineage. Low expression levels of RARgamma2 and RXRbeta remained unchanged, irrespective of differentiation pathway. Consistent with the gene expression patterns, RARalpha agonists and antagonists stimulated myelomonocytic and erythroid differentiation of FDCP-mixA4 cells, respectively. Taken together, these results suggest that erythropoiesis and granulopoiesis require diminished and enhanced RARalpha activities, respectively, which at physiological all-trans-retinoic acid (RA) concentrations may be accomplished by reciprocal effects of EPO and myelomonocytic growth factors on its expression. This hypothesis is corroborated by data showing that RA, which positively regulates RARalpha2 expression, can exert inhibitory effects on erythroid differentiation.

Potentiation of GATA-2 activity through interactions with the promyelocytic leukemia protein (PML) and the t(15;17)-generated PML-retinoic acid receptor alpha oncoprotein.

The hematopoietically expressed GATA family of transcription factors function as key regulators of blood cell fate. Among these, GATA-2 is implicated in the survival and growth of multipotential progenitors. Here we report that the promyelocytic leukemia protein (PML) can complex with GATA-2 and potentiate its transactivation capacity. The binding is mediated through interaction of the zinc finger region of GATA-2 and the B-box domain of PML. The B-box region of PML is retained in the PML-RARalpha (retinoic acid receptor alpha) fusion protein generated by the t(15;17) translocation characteristic of acute promyelocytic leukemia (APL). Consistent with this, we provide evidence that GATA-2 can physically associate with PML-RARalpha. Functional experiments further demonstrated that this interaction has the capacity to render GATA-dependent transcription inducible by retinoic acid, raising the possibility that GATA target genes may be involved in the molecular pathogenesis of APL.

B-cell commitment: Pax5 is the deciding factor.

Recent studies have identified the transcription factor Pax5 as a critical determinant of commitment to the B-lymphocyte pathway. Surprisingly, Pax5 appears to achieve this primarily through suppressing alternative haematopoietic lineage fates.

Functional and molecular analysis of hematopoietic progenitors derived from the aorta-gonad-mesonephros region of the mouse embryo.

Herein, we show that CD34, c-kit double-positive (CD34(+)c-kit(+)) cells from the aorta-gonad-mesonephros (AGM) region of the developing mouse are multipotent in vitro and can undergo both B-lymphoid and multimyeloid differentiation. Molecular analysis of individual CD34(+)c-kit(+) cells by single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) shows coactivation of erythroid (beta-globin) and myeloid (myeloperoxidase [MPO]) but not lymphoid-affiliated (CD3, Thy-1, and lambda5) genes. Additionally, most cells coexpress the stem cell-associated transcriptional regulators AML-1, PU.1, GATA-2 and Lmo2, as well as the granulocyte colony-stimulating factor receptor (G-CSF-R). These results show that the CD34(+)c-kit(+) population from the AGM represents a highly enriched source of multipotent hematopoietic cells, and suggest that limited coactivation of distinct lineage-affiliated genes is an early event in the generation of hematopoietic stem and progenitor cells during ontogeny.

A GATA-2/estrogen receptor chimera functions as a ligand-dependent negative regulator of self-renewal.

The transcription factor GATA-2 is expressed in hematopoietic stem and progenitor cells and is functionally implicated in their survival and proliferation. We have used estrogen and tamoxifen-inducible forms of GATA-2 to modulate the levels of GATA-2 in the IL-3-dependent multipotential hematopoietic progenitor cell model FDCP mix. Ligand-dependent induction of exogenous GATA-2 activity did not rescue cells deprived of IL-3 from apoptosis. However, induction of GATA-2 activity in cells cultured in IL-3 blocked factor-dependent self-renewal but not factor-dependent survival: Cells undergo cell cycle arrest and cease proliferating but do not apoptose. This was accompanied by differentiation down the monocytic and granulocytic pathways. Differentiation occurred in the presence of IL-3 and did not require addition of exogenous differentiation growth factors such as G-CSF or GM-CSF normally required to induce granulomonocytic differentiation of FDCP-mix cells. Conversely, EPO-dependent erythroid differentiation was inhibited by GATA-2 activation. These biological effects were obtained with levels of exogenous GATA-2 representing less than twofold increases over endogenous GATA-2 levels and were not observed in cells overexpressing GATA-1/ER. Similar effects on proliferation and differentiation were also observed in primary progenitor cells, freshly isolated from murine bone marrow and transduced with a GATA-2/ER-containing retrovirus. Taken together, these data suggest that threshold activities of GATA-2 in hematopoietic progenitor cells are a critical determinant in influencing self-renewal versus differentiation outcomes.

Lymphopain, a cytotoxic T and natural killer cell-associated cysteine proteinase.

Through differential screening of established human leukaemia cell lines, we have identified and molecularly cloned lymphopain, a novel cysteine proteinase of the papain family. Lymphopain exhibits a remarkably restricted cellular pattern of expression, being predominantly expressed in cytotoxic T-lymphocytes and natural killer cells. The human lymphopain locus maps to chromosome 11q13, encodes a polypeptide of 376 amino acids and is conserved in the mouse. Both human and murine forms appear more closely related to protozoan papain-like enzymes than to other mammalian members of the papain family. The cellular distribution of lymphopain expression, together with the functional demonstration of lymphopain-associated proteinase activity in vitro, is suggestive of a role for lymphopain in immune cell-mediated, cell killing.

The beta-globin LCR is not necessary for an open chromatin structure or developmentally regulated transcription of the native mouse beta-globin locus.

The murine beta-globin locus control region (LCR) was deleted from its native chromosomal location. The approximately 25 kb deletion eliminates all sequences and structures homologous to those defined as the human LCR. In differentiated ES cells and erythroleukemia cells containing the LCR-deleted chromosome, DNasel sensitivity of the beta-globin domain is established and maintained, developmental regulation of the locus is intact, and beta-like globin RNA levels are reduced 5%-25% of normal. Thus, in the native murine beta-globin locus, the LCR is necessary for normal levels of transcription, but other elements are sufficient to establish the open chromatin structure, transcription, and developmental specificity of the locus. These findings suggest a contributory rather than dominant function for the LCR in its native location.